Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Nutritional profile of rodent diets impacts experimental reproducibility in microbiome preclinical research.

The lack of reproducibility of animal experimental results between laboratories, particularly in studies investigating the microbiota, has raised concern among the scientific community. Factors such as environment, stress and sex have been identified as contributors, whereas dietary composition has received less attention. This study firstly evaluated the use of commercially available rodent diets across research institutions, with 28 different diets reported by 45 survey respondents. Secondly, highly variable ingredient, FODMAP (Fermentable Oligo-, Di-, Mono-saccharides And Polyols) and gluten content was found between different commercially available rodent diets. Finally, 40 mice were randomized to four groups, each receiving a different commercially available rodent diet, and the dietary impact on cecal microbiota, short- and branched-chain fatty acid profiles was evaluated. The gut microbiota composition differed significantly between diets and sexes, with significantly different clusters in ß-diversity. Total BCFA were highest (p = 0.01) and SCFA were lowest (p = 0.03) in mice fed a diet lower in FODMAPs and gluten. These results suggest that nutritional composition of commercially available rodent diets impact gut microbiota profiles and fermentation patterns, with major implications for the reproducibility of results across laboratories. However, further studies are required to elucidate the specific dietary factors driving these changes.

Analgesia and mouse strain influence neuromuscular plasticity in inflamed intestine.

BACKGROUND: Mouse models of inflammatory bowel disease (IBD) identify an impact on the enteric nervous system (ENS) but do not distinguish between Crohn's disease and ulcerative colitis phenotypes. In these models, analgesia is required, but its influence on different strains and disease outcomes is unknown. Therefore, changes to the ENS and intestinal smooth muscle were studied in trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) induced colitis to identify the effects of analgesia, and compared between two mouse strains. METHODS: Colitis was induced in CD1 or BALB/c mice receiving analgesia with either buprenorphine or tramadol. Euthanasia was on Day 8 (DSS) or Day 4 (TNBS). Outcomes were Disease Activity Index and cytokine assay, and quantitative histology and immunocytochemistry were used to evaluate effects of inflammation on neurons and smooth muscle. KEY RESULTS: In BALB/c mice, both models of colitis caused >2-fold increase in smooth muscle cell number. DSS caused axon proliferation without neuron loss while TNBS caused significant neuron loss and axonal damage. Buprenorphine (but not tramadol) was generally anti-inflammatory in both strains, but correlated with lethal outcomes to TNBS in BALB/c mice. CONCLUSIONS AND INFERENCES: Smooth muscle growth is common to both models of colitis. In contrast, ENS damage in TNBS is correlated with the severe response of a Crohn's disease-like phenotype, while DSS correlates with a milder, ulcerative colitis-like outcome in the deeper tissues. Analgesia with tramadol over buprenorphine is supported for mouse studies of IBD.

Chronic prenatal ethanol exposure increases adiposity and disrupts pancreatic morphology in adult guinea pig offspring.

BACKGROUND: Ethanol consumption during pregnancy can lead to a range of adverse developmental outcomes in children, termed fetal alcohol spectrum disorder (FASD). Central nervous system injury is a debilitating and widely studied manifestation of chronic prenatal ethanol exposure (CPEE). However, CPEE can also cause structural and functional deficits in metabolic pathways in offspring. OBJECTIVES AND METHODS: This study tested the hypothesis that CPEE increases whole-body adiposity and disrupts pancreatic structure in guinea pig offspring. Pregnant guinea pigs received ethanol (4 g kg(-1) maternal body weight per day) or isocaloric-sucrose/pair-feeding (control) for 5 days per week throughout gestation. RESULTS: Male and female CPEE offspring demonstrated growth restriction at birth, followed by a rapid period of catch-up growth before weaning (postnatal day (PD) 1-7). Whole-body magnetic resonance imaging (MRI) in young adult offspring (PD100-140) revealed increased visceral and subcutaneous adiposity produced by CPEE. At the time of killing (PD150-200), CPEE offspring also had increased pancreatic adipocyte area and decreased ß-cell insulin-like immunopositive area, suggesting reduced insulin production and/or secretion from pancreatic islets. CONCLUSION: CPEE causes increased adiposity and pancreatic dysmorphology in offspring, which may signify increased risk for the development of metabolic syndrome and type 2 diabetes mellitus.